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Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H + /drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs. Gram-negative bacteria can display intrinsic antibiotic resistance due to the action of tripartite efflux pumps, which include a H + /drug antiporter component. Here, the authors present a structure-function analysis of antiporter AcrB in intermediate states of the transport cycle, showing novel drug-binding sites and transport pathways.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump. Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, the authors identify pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through allosteric inhibition of its primary RND transporter.

The deployment of multidrug efflux pumps is a powerful defence mechanism for Gram-negative bacterial cells when exposed to antimicrobial agents. The major multidrug efflux transport system in Escherichia coli , AcrAB–TolC, is a tripartite system using the proton-motive force as an energy source. The polyspecific substrate-binding module AcrB uses various pathways to sequester drugs from the periplasm and outer leaflet of the inner membrane. Here we report the asymmetric AcrB structure in complex with fusidic acid at a resolution of 2.5 Å and mutational analysis of the putative fusidic acid binding site at the transmembrane domain. A groove shaped by the interface between transmembrane helix 1 (TM1) and TM2 specifically binds fusidic acid and other lipophilic carboxylated drugs. We propose that these bound drugs are actively displaced by an upward movement of TM2 towards the AcrB periplasmic porter domain in response to protonation events in the transmembrane domain. The AcrB module of the AcrAB-TolC multidrug efflux pump sequesters drugs from the periplasm and outer leaflet of the inner membrane. Here, Oswald et al . provide evidence that lipophilic carboxylated substrates bind to a groove between transmembrane helices TM1 and TM2, for further transport by an upward movement of TM2.

Acinetobacter baumannii is an opportunistic pathogen notable for multidrug resistance and environmental persistence. We characterized a clinical isolate, HKAB-1, which exhibits pronounced virulence-associated traits despite being highly susceptible to all tested antibiotics. HKAB-1 exhibited superior growth in MH2B, serum and desiccating conditions, robust biofilm formation, and active motility. Whole-genome sequencing identified two heme utilization clusters, multiple siderophore biosynthesis pathways, and other virulence-associated genes. Gene expression analysis revealed significant upregulation of heme utilization and siderophore biosynthetic gene clusters under serum exposure, indicating activation of iron uptake pathways under host-like conditions. Biofilm-associated genes, including bap, PNAG biosynthetic genes, and type IV pili components, were notably upregulated in biofilm-forming cells, supporting their role in driving the enhanced biofilm phenotype. Conversely, adeB, encoding a major RND efflux pump, was markedly downregulated, potentially explaining its drug-susceptible phenotype. Comparative genomic analysis highlighted differences in genes related to nutrient transport, metabolic pathways, and membrane biogenesis that may underpin its enhanced growth. These findings point to a potential trade-off between antibiotic resistance and virulence, underscoring the importance of monitoring antibiotic-susceptible yet highly virulent A. baumannii isolates as potential reservoirs for resistance evolution. Further investigation is warranted to elucidate the mechanisms underlying this phenotypic balance.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix–loop–helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

The existence of multiple-antibiotic-resistant strains of environmental bacteria is commonly linked to human activities. However, multiple-antibiotic-resistant strains of bacteria are also widely found in the Antarctic that has limited human activity. This study was conducted to examine the prevalence of antibiotic-resistant strains among Antarctic bacteria. Forty-five bacterial strains from Estrellas lake of King George Island and Crater lake of Deception Island, Antarctic, were exposed to 30 different antibiotics. Forty out of the 45 bacterial strains were affiliated to 12 genera, Aeromicrobium , Arthrobacter , Bacillus , Brevundimonas , Cryobacterium , Dyadobacter , Flavobacterium , Methylibium , Pedobacter , Pseudomonas , Rhodococcus , and Sphingomonas . Among the bacteria, 43 strains were resistant to at least three antibiotics, and 26 strains were resistant to 10 or more different antibiotics. Pseudomonas spp. and four unknown Microbacteriaceae bacteria were found to be resistant to majority of the antibiotics tested. Two bacteria, each from Estrellas and Crater lakes, were sensitive to all the antibiotics tested. These results indicated that Antarctic bacteria are probably the reservoirs for antibiotic resistance genes.

This report describes the isolation and characterization of bacterial isolates that produce anti-microbial compounds from one of the South Shetland Islands, King George Island, Antarctica. Of a total 2465 bacterial isolates recovered from the soil samples, six (BG5, MTC3, WEK1, WEA1, MA2 and CG21) demonstrated inhibitory effects on the growth of one or more Gram-negative or Gram-positive indicator foodborne pathogens (i.e. Escherichia coli 0157:H7, Salmonella spp., Klebsiella pneumoniae, Enterobacter cloacae, Vibrio parahaemolyticus and Bacillus cereus). Upon examination of their 16S rRNA sequences and biochemical profiles, the six Antarctic bacterial isolates were identified as Gram-negative Pedobacter cryoconitis (BG5), Pseudomonas migulae (WEK1), P. corrugata (WEA1) and Pseudomonas spp. (MTC3, MA2, and CG21). While inhibitors produced by strains BG5, MTC3 and CG21 were sensitive to protease treatment, those produced by strains WEK1, WEA1, and MA2 were insensitive to catalase, lipase, α-amylase, and protease enzymes. In addtion, the six Antarctic bacterial isolates appeared to be resistant to multiple antibiotics.